PROJECT SUMMARY This proposal focuses on histone code reader Cbx3/HP1? potential ability to regulate the stability/expression of FOXP3 (X chromosome-encoded transcription factor forkhead box P3), and whether its regulatory function can be commandeered to treat autoimmune disorders, graft-versus-host disease (GVHD) and/or prevent graft rejection. CD4+ regulatory T (Treg) cells represent a unique lineage of CD4+ T cells whose crucial function is to maintain self-tolerance and control autoimmunity. Thus, Treg cells are being exploited to treat GVHD and some autoimmune disorders. Due to its low numbers, naturally occurring Treg cells are difficult to isolate. For this reason, induced Treg (iTreg) cells are being used in the clinic. However, the instability of FOXP3 expression in Treg cells poses a serious potential limitation. Therefore, the challenge is to identify novel means to stabilize FOXP3 expression. FOXP3 stability/expression is regulated in part by three conserved noncoding sequence (CNS1/2/3) elements found in the first intron of Foxp3. Of these, CNS2 controls the stability of FOXP3 expression through CNS2 DNA modification (methylation or demethylation) status. DNA methylation is controlled by a balance between DNA methyltransferases (DNMTs) and DNA demethylases that act in concert with transcription factors and accessory proteins to regulate gene expression. Genetic deletion of Dnmt1 leads to enhanced FOXP3 expression suggesting that DNMT1 limits FOXP3 expression. How DNMT1 activity is regulated in Treg cells is still unknown. An attractive target is Cbx3/HP1? shown to interact with methyl groups of histones H3 at lysine 9 (H3K9Me3) and DNMT1. Our preliminary results demonstrate that Cbx3/HP1?-deficient iTreg cells express more FOXP3, both protein and transcripts. We propose that in normal CD4+ Treg cells, Cbx3/HP1? restrains FOXP3 expression. Mechanistically, Cbx3/HP1? occupies the Foxp3 locus via its ability to interact with histones H3K9m3 as well as DNMT1 to maintain a fully methylated and repressed Foxp3 locus. In the absence or decreased level of Cbx3/HP1?, methylation of Foxp3 is lost or reduced resulting in FOXP3 sustained and stable expression. Thus, Cbx3/HP1?- deficient iTreg cells would be more stable and could be used to treat immune disorders and/or GVHD. If successful, our proposal will reveal that the histone code reader Cbx3/HP1? regulates Foxp3 stability/expression. Our findings will contribute to understanding the complex epigenetic regulation of Foxp3 stability/expression and Treg development. Our results will provide novel means to manipulate the epigenome of CD4+ T cells to generate stable iTregs with sustained Foxp3 expression to treat autoimmune disorders, GVHD and/or prevent chronic graft rejection in humans.